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WO2006002102A2 - Composes spirocycliques utiles en tant que modulateurs de la fonction du recepteur de l'hormone nucleaire - Google Patents

Composes spirocycliques utiles en tant que modulateurs de la fonction du recepteur de l'hormone nucleaire Download PDF

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WO2006002102A2
WO2006002102A2 PCT/US2005/021823 US2005021823W WO2006002102A2 WO 2006002102 A2 WO2006002102 A2 WO 2006002102A2 US 2005021823 W US2005021823 W US 2005021823W WO 2006002102 A2 WO2006002102 A2 WO 2006002102A2
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substituted
alkyl
aryl
cycloalkyl
cycloalkylalkyl
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WO2006002102A3 (fr
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James Aaron Balog
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Bristol Myers Squibb Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/20—Spiro-condensed ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to spirocyclic compounds, to methods of using such compounds in the treatment of nuclear hormone receptor-associated conditions such as cancer, and to pharmaceutical compositions containing such compounds.
  • Nuclear hormone receptors constitute a large super-family of ligand-dependent and sequence-specific transcription factors. Members of this family influence transcription either directly, through specific binding to the promoter target genes (Evans, in Science 240: 889-895 (1988)), or indirectly, via protein-protein interactions with other transcription factors (Jonat et al., Cell 62: 1189-1204 (1990), Schuele et al., Cell 62: 1217-1226 (1990), and Yang- Yen et al., Cell 62: 1205-1215 (1990)).
  • the nuclear hormone receptor super-family (also known in the art as the "steroid/thyroid hormone receptor super-family”) includes receptors for a variety of hydrophobic ligands, including Cortisol, aldosterone, estrogen, progesterone, testosterone, vitamine D3, thyroid hormone, and retinoic acid (Evans, 1988, supra).
  • the super-family contains a number of proteins that have no known ligands, termed orphan nuclear hormone receptors (Mangelsdorf et al., Cell 83: 835-839 (1995), O'Malley et al., MoI. Endocrinol. 10: 1293 (1996), Enmark et al., MoI. Endocrinol.
  • the conventional nuclear hormone receptors are generally transactivators in the presence of ligand, and can either be active repressors or transcriptionally inert in the absence of ligand. Some of the orphan receptors behave as if they are transcriptionally inert in the absence of ligand. Others, however, behave as either constitutive activators or repressors. These orphan nuclear hormone receptors are either under the control of ubiquitous ligands that have not been identified, or do not need to bind ligand to exert these activities.
  • the nuclear hormone receptors have a modular structure, being comprised of three distinct domains: an N-terminal domain of variable size containing a transcriptional activation function AF-I, a highly conserved DNA binding domain and a moderately conserved ligand-binding domain.
  • the ligand-binding domain is not only responsible for binding the specific ligand but also contains a transcriptional activation function called AF-2 and a dimerisation domain (Wurtz et al., Nature Stmc. Biol. 3, 87-94 (1996), Parker et al., Nature Struc. Biol. 3, 113-115 (1996) and Kumar et al., Steroids 64, 310-319 (1999)).
  • SB-NHR' s The steroid binding nuclear hormone receptors (SB-NHR' s) comprise a sub-family of nuclear hormone receptors. These receptors are related in that they share a stronger sequence homology to one another, particularly in the ligand binding domain (LBD), than to the other members of the NHR super-family (Evans, 1988, supra) and they all utilize steroid based ligands.
  • NHR' s are the androgen receptor (AR), the estrogen receptor (ER), the progesterone receptor (PR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), the aldosterone receptor (ALDR), and the steroid and xenobiotic receptor (SXR) (Evans et al., WO 99/35246).
  • AR androgen receptor
  • ER estrogen receptor
  • PR progesterone receptor
  • GR glucocorticoid receptor
  • MR mineralocorticoid receptor
  • ADR aldosterone receptor
  • SXR steroid and xenobiotic receptor
  • Examples of some of the steroid based ligands utilized by members of the SB-NHR' s include Cortisol, aldosterone, estrogen, progesterone, testosterone, and dihydrotestosterone. Specificity of a particular steroid based ligand for one SB-NHR versus another is obtained by differential substitution about the steroid core. High affinity binding to a particular SB-NHR, coupled with high level specificity for that particular SB-NHR, can be achieved with only minor structural changes about the steroid core (e.g., Waller et al., Toxicol. Appl. Pharmacol. 137, 219-227 (1996) and Mekenyan et al., Environ. ScL Technol.
  • RU486 is an example of a synthetic agonist of the PR, which is utilized as a birth control agent (Vegeto et al., Cell 69: 703-713 (1992)), and Flutamide is an example of an antagonist of the AR, which is utilized for the treatment of prostate cancer (Neri et al, Endo. 91, 427-437 (1972)).
  • Tamoxifen is an example of a tissues specific modulator of the ER function, that is used in the treatment of breast cancer (Smigel, /. Natl. Cancer Inst. 90, 647-648 (1998)). Tamoxifen can function as an antagonist of the ER in breast tissue while acting as an agonist of the ER in bone (Grese et al., Proc. Natl. Acad. ScL USA 94, 14105-14110 (1997)). Because of the tissue selective effects seen for Tamoxifen, this agent and agents like it are referred to as "partial-agonist" or partial-antagonist".
  • non-endogenous ligands for NHR' s can be obtained from food sources (Regal et al., Proc. Soc. Exp. Biol. Med. 223, 372- 378 (2000) and Hempstock et al., J. Med. Food 2, 267-269 (1999)).
  • the flavanoid phytoestrogens are an example of a non-natural ligand for SB-NHR' s that are readily obtained from a food source such as soy (Quella et al., J. Clin. Oncol. 18, 1068-1074 (2000) and Banz et al., J. Med. Food 2, 271-273 (1999)).
  • non-natural ligands can be synthetically engineered to serve as modulators of the function of NHR' s.
  • engineering of an non-natural ligand can include the identification of a core structure which mimics the natural steroid core system.
  • spirocyclic compounds of the present invention comprise a core which serves as a steroid mimic, and are useful as modulators of the function of steroid binding nuclear hormone receptors, as well as other NHR as described herein.
  • the present invention provides spirocyclic compounds of the following formula (I) and salts thereof:
  • G is CH or N;
  • X and Y are independently selected from H, CN, NO 2 , F, Cl, Br, I, CF 3 , CF 2 CF 3 , R 1 , OR 1 , COR 1 , CONR 1 R 1' , and/or NR 1 R 1' ;
  • a pharmaceutical composition comprising at least one of the spirocyclic compounds of formula (I), a pharmaceutically acceptable salt, solvate, clathrate, hydrate, or prodrug thereof; and a pharmaceutically acceptable carrier.
  • a method of modulating the function of a nuclear hormone receptor comprising administrating to a subject in need of treatment thereof, an effect amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, clathrate, hydrate, or prodrug thereof.
  • a method of treating a condition or disorder comprising administering to a mammalian species in need thereof a therapeutically effective amount of a compound of formula (I), a pharmaceutically acceptable salt, solvate, clathrate, hydrate, or prodrug thereof.
  • alkyl and “alk” refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms.
  • Exemplary such groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4- dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like.
  • “Substituted alkyl” refers to an alkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
  • alkenyl refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include ethenyl or allyl.
  • Substituted alkenyl refers to an alkenyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
  • substituents include, but are not limited to, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents.
  • cycloalkyl refers to a fully saturated or partially saturated cyclic hydrocarbon group containing from 1 to 4 rings and 3 to 8 carbons per ring.
  • Exemplary fully saturated cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • Exemplary partially saturated cycloalkyl groups include cyclobutenyl, cyclopentenyl, and cyclohexenyl.
  • “Substituted cycloalkyl” refers to a cycloalkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
  • Exemplary substituents include, but are not limited to, nitro, cyano, alkyl or substituted alkyl, as well as those groups recited above as exemplary alkyl substituents.
  • Exemplary substituents also include spiro-attached or fused cyclic substituents, especially cycloalkenyl or substituted cycloalkenyl.
  • alkoxy or “alkylthio” refer to an alkyl group as described- above bonded through an oxygen linkage (-O-) or a sulfur linkage (-S-), respectively.
  • substituted alkoxy or “substituted alkylthio” refer to a substituted alkyl group as described above bonded through an oxygen or sulfur linkage, respectively.
  • alkoxycarbonyl refers to an alkoxy group bonded through a carbonyl group.
  • alkylcarbonyl refers to an alkyl group bonded through a carbonyl group.
  • alkylcarbonyloxy refers to an alkylcarbonyl group bonded through an oxygen linkage.
  • arylalkyl refers to cyclic, aromatic hydrocarbon groups which have 1 to 5 aromatic rings, especially monocyclic or bicyclic groups such as phen
  • the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl), or fused (e.g., naphthyl, phenanthrenyl and the like).
  • substituted aryl refers to an aryl group substituted by one or more substituents, preferably 1, 2, 3, 4, or 5 substituents, at any point of attachment.
  • substituents also include fused cyclic substituents, such as heterocyclo, cycloalkenyl, substituted heterocyclo, and cycloalkenyl groups (e.g., thereby forming a fluoroenyl, tetrahydronapthalenyl, or dihydroindenyl group).
  • Carbamoyl refers to the group -CONH- which is bonded on one end to the remainder of the molecule and on the other to hydrogen or an organic moiety (such as alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, alkylcarbonyl, hydroxyl, and substituted nitrogen).
  • Carbamoyl refers to the group -CONH- which is bonded on one end to the remainder of the molecule and on the other to hydrogen or an organic moiety (such as those listed above).
  • “Carbamate” refers to the group -OCO-NH- which is bonded on one end to the remainder of the molecule and on the other to hydrogen or an organic moiety (such as those listed above).
  • “Urea” refers to the group -NH-CO-NH- which is bonded on one end to the remainder of the molecule and on the other to hydrogen or an organic moiety (such as those listed above).
  • “Substituted carbamoyl,” “substituted carbamate,” “substituted urea”, and “substituted amidinyl” refer to carbamoyl, carbamate, urea., and amidinyl groups, respectively, as described above in which one more of the hydrogen groups are replaced by an organic moiety (such as those listed above).
  • heterocycle refers to fully saturated, or partially or fully unsaturated, including aromatic (i.e., “heteroaryl”) cyclic groups (for example, 3 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16 membered tricyclic ring systems) which have at least one heteroatom in at least one carbon atom-containing ring.
  • aromatic i.e., "heteroaryl”
  • cyclic groups for example, 3 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16 membered tricyclic ring systems
  • Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
  • heteroarylium refers to a heteroaryl group bearing a quaternary nitrogen atom and thus a positive charge.
  • the heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system.
  • Exemplary monocyclic heterocyclic groups include ethylene oxide, azetidinyl, pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxo ⁇ iperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, hexahydrodiazepinyl, 4-piperidonyl
  • bicyclic heterocyclic groups include indolyl, isoindolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl], or furo[2,3-b]pyridinyl), dihydrobenzodioxinyl, dihydrodioxidobenzo
  • Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl, and the like.
  • "Substituted heterocycle,” “substituted heterocyclic,” and “substituted heterocyclo” refer to heterocycle, heterocyclic, or heterocyclo groups substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
  • quaternary nitrogen refers to a tetravalent positively charged nitrogen atom including, for example, the positively charged nitrogen in a tetraalkylammonium group (e.g., tetramethylammonium or N-methylpyridinium), the positively charged nitrogen in protonated ammonium species (e.g., trimethyl- hydroammonium or N-hydropyridinium), the positively charged nitrogen in amine N- oxides (e.g., N-methyl-morpholine-N-oxide or pyridine-N-oxide), and the positively charged nitrogen in an N-amino-ammonium group (e.g., N-aminopyridinium).
  • a tetraalkylammonium group e.g., tetramethylammonium or N-methylpyridinium
  • protonated ammonium species e.g., trimethyl- hydroammonium or N-hydropyridinium
  • the positively charged nitrogen in amine N- oxides
  • halogen or “halo” refer to chlorine, bromine, fluorine, and iodine.
  • hydroxylamine and “hydroxylamide” refer to the groups OH- NH- and OH-NH-CO-, respectively.
  • protecting groups for the methods and compounds described herein include, without limitation, those described in standard textbooks, such as Greene, T. W. et al., Protective Groups in Organic Synthesis, Wiley, N. Y. (1991).
  • any heteroatom with unsatisfied valences is assumed to have hydrogen atoms sufficient to satisfy the valences.
  • Carboxylate anion refers to a negatively charged group -COO " .
  • the spirocyclic compounds of formula I may form salts which are also within the scope of this invention. Reference to a spirocyclic compound of the formula I herein is understood to include reference to salts thereof, unless otherwise indicated.
  • the term "salt(s)" denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases.
  • salts when a compound of formula I contains both a basic moiety, such as but not limited to a pyridine or imidazole, and an acidic moiety such as but not limited to a carboxylic acid, zwitterions may be formed and are included within the term "salt(s)" as used herein.
  • Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation.
  • Salts of the spirocyclic compounds of the formula I may be formed, for example, by reacting a spirocyclic compound of formula I with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
  • the spirocyclic compounds of formula I may contain a basic moiety, such as but not limited to an amine or a pyridine or imidazole ring, may form salts with a variety of organic and inorganic acids.
  • Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, hydroxyethanesulfonates (e.g., 2-hydroxyethanesulfonates), lactates, maleates, methanesulfonates, naphthalenesulfonates (e
  • the spirocyclic compounds of formula I may contain an acidic moiety, such but not limited to a carboxylic acid, may form salts with a variety of organic and inorganic bases.
  • Exemplary basic salts include ammonium salts; alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N- bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D- glycamides, t-butyl amines; and salts with amino acids such as arginine, lysine, and the like.
  • Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g. methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g. decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.
  • lower alkyl halides e.g. methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides
  • dialkyl sulfates e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates
  • Prodrugs and solvates of the spirocyclic compounds of the invention are also contemplated herein.
  • the term "prodrug” as employed herein denotes a compound which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of the formula I, or a salt and/or solvate thereof.
  • Solvates of the spirocyclic compounds of formula I include, for example, hydrates.
  • All stereoisomers of the present compounds for example, those which may exist due to asymmetric carbons on various substituents, including enantiomeric forms and diastereomeric forms, are contemplated within the scope of this invention.
  • Individual stereoisomers of the spirocyclic compounds of the invention may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers.
  • the chiral centers of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations.
  • the racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography.
  • the individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
  • All configurational isomers of the spirocyclic compounds of the present invention are contemplated, either in admixture, or in pure or substantially pure form.
  • the preferred configuration can be a function of the particular compound and its preferred activity. Separation of configurational isomers can be achieved by any suitable method, such as column chromatography.
  • groups and substituents thereof may be chosen to provide stable moieties and compounds.
  • Embodiments indicated herein as exemplary or preferred are intended to be illustrative and not limiting.
  • the spirocyclic compound of formula (I) has a structure in which: X and Y are independently selected from H, CN, NO 2 , F, Cl, Br, I, CH 3 , CH 2 CH 3 , CF 3 , and/or CF 2 CF 3 ; Z is selected from H, CN, F, Cl, Br, I, CH 3 , CH 2 CH 3 , CF 3 , or CF 2 CF 3 ; G, Q 1 , Q 2 , Q 3 , Q 4 , R 1 , R 1 , R 2 , and R 4 are defined hereinabove, and provided that: (1) when G is CH and Z is H, then X and Y cannot be fused to form an unsubstituted aryl ring; (2) when G is CH and X is H, then Y and Z cannot be fused to form an unsubstituted aryl ring; (3) when G is CH, Y is CH 3 , and Z is H
  • the spirocyclic compound of formula (I) has a structure in which G is N.
  • the spirocyclic compound of this embodiment has the formula (HI):
  • the spirocyclic compound of formula I has a structure in which Q 1 , Q 2 , Q 3 , and Q 4 are independently selected from H, methyl, ethyl, propyl, and/or CH 2 CF 3 .
  • the spirocyclic compound of formula I has at least one R 4 substituent comprising a piperazinyl group or a substituted piperazinyl group. Examples of suitable R 4 substituents include, but are not limited to, the piperazinyl groups and the substituted piperazinyl groups represented by formula (IV):
  • spirocyclic compounds of the present invention may be prepared by methods such as those illustrated in the following Schemes I and II. Solvents, temperatures, pressures, and other reaction conditions may readily be selected by one of ordinary skill in the art. Starting materials are commercially available or readily prepared by one skilled in the art. Combinatorial techniques may be employed in the preparation of compounds, for example, where the intermediates possess groups suitable for these techniques. Scheme I
  • Scheme I demonstrates a possible synthesis of substituted spirocyclic anhydrides VI.
  • the methyl enolate II can be treated with an allyl organometallic such as allyl magnesiumbromide in the presence of copper (I) iodide to give the product III via 1,4-conjugate addition (Journal of Organic Chemistry, 51(10), 1745-53, 1986).
  • the ester can then be alkylated in the alpha position by successive treatment with a base such as LDA and an alkylating agent such as methyl iodide.
  • the alkylating agent can vary widely as known by one skilled in the art. This process can then be repeated to give compounds of general formula IV.
  • the spirocyclic anhydride of formula VI can be coupled with the amine of formula VII under a variety of dehydrating conditions to give a compound of formula VHI which is a compound of formula I, as exemplified in U.S. Patent Publication 2002/0058290 and 2003/0181728. The conditions shown have been successful for compounds presented herein.
  • the spirocyclic compounds of the present invention are useful for modulating the function of nuclear hormone receptors (NHR), and include compounds which are, for example, agonists, partial agonists, antagonists, or partial antagonists of the androgen receptor (AR), the estrogen receptor (ER), the progesterone receptor (PR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), the steroid and xenobiotic receptor (SXR), other steroid binding NHR's, the Orphan receptors, or other NHR's. Selective modulation of one such NHR relative to others within the NHR family is preferred.
  • NHR nuclear hormone receptors
  • AR androgen receptor
  • ER estrogen receptor
  • PR progesterone receptor
  • GR glucocorticoid receptor
  • MR mineralocorticoid receptor
  • SXR steroid and xenobiotic receptor
  • Selective modulation of one such NHR relative to others within the NHR family is preferred.
  • NHR-associated condition denotes a condition or disorder which can be treated by modulating the function of a NHR in a subject, wherein treatment comprises prevention, partial alleviation, or cure of the condition or disorder. Modulation may occur locally, for example, within certain tissues of the subject, or more extensively throughout a subject being treated for such a condition disorder.
  • the spirocyclic compounds of the present invention are useful for the treatment of a variety of conditions and disorders including, but not limited to, those described following: [0052]
  • the spirocyclic compounds of formula I can be applied as agonists, partial agonists, antagonists, or partial antagonists of the estrogen receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the estrogen receptor pathway.
  • Applications of said compounds include but are not limited to: osteoporosis, hot flushes, vaginal dryness, prostate cancer, breast cancer, endometrial cancer, cancers expressing the estrogen receptor such as the aforementioned cancers and others, contraception, pregnancy termination, menopause, amennoreahea, and dysmennoreahea.
  • the spirocyclic compounds of formula I can be applied as agonists, partial agonists, antagonists, or partial antagonists of the progesterone receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the progesterone receptor pathway.
  • Applications of said compounds include but are not limited to: breast cancer, other cancers containing the progesterone receptor, endometriosis, cachexia, contraception, menopause, cyclesynchrony, meniginoma, dysmennoreahea, fibroids, pregnancy termination, labor induction, and osteoporosis.
  • the spirocyclic compounds of formula I can be applied as agonists, partial agonists, antagonists, or partial antagonists of the glucocorticoid receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the glucocorticoid receptor pathway.
  • Applications of said compounds include but are not limited to: inflammatory diseases, autoimmune diseases, prostate cancer, breast cancer, Alzheimer's disease, psychotic disorders, drug dependence, non-insulin dependent Diabetes Mellitus, and as dopamine receptor blocking agents or otherwise as agents for the treatment of dopamine receptor mediated disorders.
  • Glucocorticoid receptor AP-I (“GR AP-I”) inhibitors can be used as anti ⁇ inflammatory and immunosuppressive agents, for example, to treat a wide variety of inflammatory and autoimmune diseases. These diseases include without limitation: rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, asthma, chronic obstructive pulmonary disease, prevention of transplant rejection, multiple sclerosis, and psoriasis, among others.
  • GR AP-I inhibitors of the present invention can be employed together with known GR AP-I inhibitors, such as the steroid prednisone (which is used to treat the above diseases).
  • the spirocyclic compounds of formula I can be applied as agonists, partial agonists, antagonists, or partial antagonists of the mineralocorticoid receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the mineralocorticoid receptor pathway. Applications of said compounds include but are not limited to: drug withdrawal syndrome and inflammatory diseases.
  • the spirocyclic compounds of formula I can be applied as agonists, partial agonists, antagonists, or partial antagonists of the aldosterone receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the aldosterone receptor pathway.
  • One application of said compounds includes but is not limited to: congestive heart failure.
  • the spirocyclic compounds of formula I can be applied as agonists, partial agonists, antagonists, or partial antagonists of the androgen receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the androgen receptor pathway.
  • inventions include but are not limited to: hirsutism, acne, seborrhea, Alzheimer's disease, androgenic alopecia, hypogonadism, hyperpilosity, benign prostate hypertrophia, adenomas and neoplasies of the prostate (such as advanced metastatic prostate cancer), treatment of benign or malignant tumor cells containing the androgen receptor such as is the case for breast, brain, skin, ovarian, bladder, lymphatic, liver and kidney cancers, pancreatic cancers, modulation of VCAM expression and applications therein for the treatment of heart disease, inflammation and immune modulations, modulation of VEGF expression and the applications therein for use as antiangiogenic agents, osteoporosis, suppressing spermatogenesis, libido, cachexia, endometriosis, polycystic ovary syndrome, anorexia, androgen supplement for age related decreased testosterone levels in men, male menopause, male hormone replacement, male and female sexual dysfunction, and inhibition of muscular at
  • pan AR modulation is contemplated, with prostate selective AR modulation ("SARM”) being particularly preferred, such as for the treatment of early stage prostate cancers.
  • SARM prostate selective AR modulation
  • the spirocyclic compounds of formula I can be applied as (preferably, selective) antagonists of the mutated androgen receptor, for example, found in many tumor lines. Examples of such mutants are those found in representative prostate tumor cell lines such as LNCap, (T877A mutation, Biophys. Acta, 187, 1052 (1990)), PCa2b, (L701H & T877A mutations, /. Urol, 162, 2192 (1999)) and CWR22, (H874Y mutation, MoI. Endo., 11, 450 (1997)).
  • the spirocyclic compounds of formula I can be applied as agonists, partial agonists, antagonists, or partial antagonists of the steroid and xenobiotic receptor, preferably selectively to that receptor, in an array of medical conditions which involve modulation of the steroid and xenobiotic receptor pathway.
  • Applications of said compounds include but are not limited to: treatment of disregulation of cholesterol homeostasis, attenuation of metabolism of pharmaceutical agents by co- administration of an agent (compound of the present invention) which modulates the P450 regulator effects of SXR.
  • NHR NHR due to strong sequence homology to other NHR
  • Orphan receptors demonstrate strong sequence homology to other NHR
  • compounds of formula I include those which serve as modulators of the function of the Orphan NHR.
  • Orphan receptors that are modulated by NHR modulators such as compounds within the scope of formula I are exemplified, but not limited to, those listed in Table 1.
  • Exemplary therapeutic applications of modulators of said Orphan receptors are also listed in Table 1, but are not limited to the examples therein.
  • the present invention thus provides methods for the treatment of NHR- associated conditions, comprising the step of administering to a subject in need thereof at least one spirocyclic compound of formula I in an amount effective therefor.
  • Other therapeutic agents such as those described below may be employed with the inventive compounds in the present methods (for example, separately, or formulated together as a fixed dose).
  • such other therapeutic agent(s) can be administered prior to, simultaneously with, or following the administration of the spirocyclic compound(s) of the present invention.
  • the present invention also provides pharmaceutical compositions comprising at least one of the spirocyclic compounds of the formula I capable of treating a NHR-associated condition in an amount effective therefor, and a pharmaceutically acceptable carrier (vehicle or diluent).
  • a pharmaceutically acceptable carrier vehicle or diluent.
  • the compositions of the present invention can contain other therapeutic agents as described below, and can be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.
  • the spirocyclic compounds of the present invention are, without limitation as to their mechanism of action, useful in treating any of the conditions or disorders listed or described herein such as inflammatory diseases or cancers, or other proliferate diseases, and in compositions for treating such conditions or disorders.
  • the present compounds have therapeutic utility in the modulation of immune cell activation/proliferation, e.g., as competitive inhibitors of intercellular ligand/receptor binding reactions involving CAMs (Cellular Adhesion Molecules) and Leukointegrins.
  • the present compounds modulate LFA-ICAM 1, and are particularly useful as LFA-ICAM 1 antagonists, and in the treatment of all conditions associated with LFA-ICAM 1 such as immunological disorders.
  • Preferred utilities for the present compounds include, but are not limited to: inflammatory conditions such as those resulting from a response of the non-specific immune system in a mammal (e.g., adult respiratory distress syndrome, shock, oxygen toxicity, multiple organ injury syndrome secondary to septicemia, multiple organ injury syndrome secondary to trauma, reperfusion injury of tissue due to cardiopulmonary bypass, myocardial infarction or use with thrombolysis agents, acute glomerulonephritis, vasculitis, reactive arthritis, dermatosis with acute inflammatory components, stroke, thermal injury, hemodialysis, leukapheresis, ulcerative colitis, necrotizing enterocolitis and granulocyte transfusion associated syndrome) and conditions resulting from a response of the specific immune system in a mammal (e.g., psoriasis,
  • the present compounds can be used in treating asthma or as an adjunct to minimize toxicity with cytokine therapy in the treatment of cancers.
  • the present compounds can be employed in the treatment of all diseases currently treatable through steroid therapy.
  • the present compounds may be employed for the treatment of these and other disorders alone or with other immunosuppressive or antiinflammatory agents.
  • a compound of the formula I can be administered prior to the onset of inflammation (so as to suppress an anticipated inflammation) or after the initiation of inflammation.
  • the immunosupressive compound(s) are preferably provided in advance of any inflammatory response or symptom (for example, prior to, at, or shortly after the time of an organ or tissue transplant but in advance of any symptoms or organ rejection).
  • the prophylactic administration of a compound of the formula I prevents or attenuates any subsequent inflammatory response (such as, for example, rejection of a transplanted organ or tissue, etc.)
  • Administration of a spirocyclic compound of the formula I attenuates any actual inflammation (such as, for example, the rejection of a transplanted organ or tissue).
  • the spirocyclic compounds of the formula I can be administered for any of the uses described herein by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; bucally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally, including administration to the nasal membranes, such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories; in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents.
  • suitable means for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; bucally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection or
  • the present compounds can, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by the use of suitable pharmaceutical compositions comprising the present compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.
  • the present compounds can also be administered liposomally.
  • compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art.
  • the spirocyclic compounds of formula I can also be delivered through the oral cavity by sublingual and/or buccal administration.
  • Molded tablets, compressed tablets or freeze-dried tablets are exemplary forms which may be used.
  • Exemplary compositions include those formulating the present compound(s) with fast dissolving diluents such as mannitol, lactose, sucrose and/or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as celluloses (avicel) or polyethylene glycols (PEG).
  • Such formulations can also include an excipient to aid mucosal adhesion such as hydroxy propyl cellulose (HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleic anhydride copolymer (e.g., Gantrez), and agents to control release such as polyacrylic copolymer (e.g. CarbopolTM 934 polymer, B.F. Goodrich Co., NY).
  • HPC hydroxy propyl cellulose
  • HPMC hydroxy propyl methyl cellulose
  • SCMC sodium carboxy methyl cellulose
  • maleic anhydride copolymer e.g., Gantrez
  • agents to control release such as polyacrylic copolymer (e.g. CarbopolTM 934 polymer, B.F. Goodrich Co., NY).
  • Lubricants, glidants, flavors, coloring agents and stabilizers may also be added for ease of fabrication and use.
  • compositions for nasal aerosol or inhalation administration include solutions in saline which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
  • compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • compositions for rectal administration include suppositories which can contain, for example, a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
  • exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).
  • the effective amount of the spirocyclic compound of the present invention can be determined by one of ordinary skill in the art, and includes exemplary dosage amounts for an adult human of from about 1 to 100 (for example, 15) mg/kg of body weight of active compound per day, which can be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. It will be understood that the specific dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.
  • Preferred subjects for treatment include animals, most preferably mammalian species such as humans, and domestic animals such as dogs, cats and the like, subject to NHR-associated conditions.
  • the spirocyclic compounds of the present invention can be employed alone or in combination with each other and/or other suitable therapeutic agents useful in the treatment of NHR-associated conditions.
  • the spirocyclic compounds of the present invention can be administered either alone or in combination with other anti-cancer and cytotoxic agents and treatments useful in the treatment of cancer or other proliferative diseases, for example, where the second drug has the same or different mechanism of action than the present compounds of formula I.
  • Examples of classes of anti-cancer and cytotoxic agents useful in combination with the present compounds include but are not limited to: alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, and triazenes; antimetabolites such as folate antagonists, purine analogues, and pyrimidine analogues; antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin, and plicamycin; enzymes such as L-asparaginase; farnesyl-protein transferase inhibitors; 5 ⁇ reductase inhibitors; inhibitors of 17 ⁇ -hydroxy steroid dehydrogenase type 3; hormonal agents such as glucocorticoids, estrogens/ antiestrogens, androgens/ antiandrogens, progestins, and luteinizing hormone-releasing hormone antagonists, octreotide acetate; microtubule-dis
  • spirocyclic compounds of the invention may also be used in conjunction with radiation therapy.
  • Representative examples of these classes of anti-cancer and cytotoxic agents include but are not limited to mechlorethamine hydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan, carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine, methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D, safracins, saframycins, quinocarcins, discodermolides, vincristine, vinblastine, vin
  • Preferred member of these classes include, but are not limited to, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C, ecteinascidin 743, or porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine and leurosine.
  • anticancer and other cytotoxic agents include the following: epothilone derivatives as found in German Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO 99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO 99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO 99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclin dependent kinase inhibitors as found in WO 99/24416 (see also U.S.
  • NHR modulators including, but not limited to, those of present invention
  • AR modulators including, but not limited to, those of present invention
  • ER modulators with LHRH modulators
  • surgical castration especially in the treatment of cancer.
  • the spirocyclic compounds of the present invention can also be formulated or co- administered with other therapeutic agents that are selected for their particular usefulness in administering therapies associated with the aforementioned conditions.
  • the spirocyclic compounds of the invention may be formulated with agents to prevent nausea, hypersensitivity and gastric irritation, such as antiemetics, and H 1 and H 2 antihistaminics.
  • the spirocyclic compounds of this invention are most preferably used alone or in combination with anti-cancer treatments such as radiation therapy and/or with cytostatic and/or cytotoxic agents, such as, but not limited to, DNA interactive agents, such as cisplatin or doxorubicin; inhibitors of farnesyl protein transferase, such as those described in U.S. Patent No.
  • topoisomerase II inhibitors such as etoposide
  • topoisomerase I inhibitors such as CPT-11 or topotecan
  • tubulin stabilizing agents such as paclitaxel, docetaxel, other taxanes, or epothilones
  • hormonal agents such as tamoxifen
  • thymidilate synthase inhibitors such as 5-fluorouracil
  • antimetabolites such as methoxtrexate
  • antiangiogenic agents such as angiostatin, ZD6474, ZD6126 and comberstatin A2
  • kinase inhibitors such as her2 specific antibodies, Iressa and CDK inhibitors
  • histone deacetylase inhibitors such as CI-994 and MS-27-275.
  • Such compounds may also be combined with agents which suppress the production of circulating testosterone such as LHRH agonists, antagonists, or with surgical castration.
  • known therapies for advanced metastatic prostate cancer include "complete androgen ablation therapy" wherein tumor growth is inhibited by controlling the supply of androgen to the prostate tissues via chemical castration (castration serves to inhibit the production of circulating testosterone (T) and dihydrotestosterone (DHT)) followed by the administration of androgen receptor (AR) antagonists (which inhibit the function T/DHT derived from the conversion of circulating androgen precursors to T/DHT by the prostate tissue).
  • the spirocyclic compounds of the present invention can be employed as AR antagonists in complete ablation therapy, alone or in combination with other AR antagonists such as Flutamide, Casodex, Nilutamide, or Cyproterone acetate.
  • the spirocyclic compounds of the present invention may be employed adjuvant to surgery.
  • Another application of the present compounds is in combination with antibody therapy such as but not limited to antibody therapy against PSCA.
  • An additional application is in concert with vaccine / immune modulating agents for the treatment of cancer.
  • the above other therapeutic agents when employed in combination with the spirocyclic compounds of the present invention, can be used, for example, in those amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.
  • the following assays can be employed in ascertaining the activity of the spirocyclic compounds of formula I as a NHR modulator. Preferred are those compounds with an activity greater than 20 ⁇ m for binding or transactivation in any of these assays.
  • the AR modulator activity of various spirocyclic compounds of the present invention can be determined by utilizing the transactivation assay and standard AR binding assays as described following. TRANSACTIVATION ASSAYS
  • the spirocyclic compounds of the present invention are tested in transactivation assays of a transfected reporter construct and using the endogenous androgen receptor of the host cells.
  • the transactivation assay provides a method for identifying functional agonists and partial agonists that mimic, or antagonists that inhibit, the effect of native hormones, in this case, dihydrotestosterone (DHT).
  • DHT dihydrotestosterone
  • This assay can be used to predict in vivo activity as there is a good correlation in both series of data. See, e.g. T. Berger et al., /. Steroid Biochem. Molec. Biol. 773 (1992), the disclosure of which is herein incorporated by reference.
  • a reporter plasmid is introduced by transfection (a procedure to induce cells to take foreign genes) into the respective cells.
  • This reporter plasmid comprising the cDNA for a reporter protein, such as secreted alkaline phosphatase (SEAP), is controlled by prostate specific antigen (PSA) upstream sequences containing androgen response elements (AREs).
  • PSA prostate specific antigen
  • AREs androgen response elements
  • the transactivation assay is carried out in the presence of constant concentration of the natural AR hormone (DHT) known to induce a defined reporter signal. Increasing concentrations of a suspected antagonist will decrease the reporter signal (e.g., SEAP production). On the other hand, exposing the transfected cells to increasing concentrations of a suspected agonist will increase the production of the reporter signal.
  • DHT natural AR hormone
  • LNCaP and MDA 453 cells are obtained from the American Type Culture Collection (Rockville, MD), and maintained in RPMI 1640 or DMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco) respectively. The respective cells are transiently transfected by electroporation according to the optimized procedure described by Heiser, 130 Methods MoI.
  • the reporter plasmid is constructed as follows: commercial human placental genomic DNA is used to generate by Polymerase Cycle Reaction (PCR) a fragment containing the BgIII site (position 5284) and the Hind DI site at position 5831 of the human prostate specific antigen promoter (Accession # U37672), Schuur, et al., J. Biol. Chem., 271 (12): 7043-51 (1996). This fragment is subcloned into the pSEAP2/basic (Clontech) previously digested with Bgi ⁇ and HindDI to generate the pSEAP2/PSA540 construct.
  • PCR Polymerase Cycle Reaction
  • a fragment bearing the fragment of human PSA upstream sequence between positions -5322 and -3873 is amplified by PCR from human placental genomic DNA.
  • a Xhol and a Bgi ⁇ sites are introduced with the primers.
  • the resulting fragment is subcloned into pSEAP2/PSA540 digested with Xhol and BgIII respectively, to generate the pSEAP2/PSA540/Enhancer construct.
  • LNCaP and MDA 453 cells are collected in media containing 10% charcoal stripped FBS.
  • Each cell suspension is distributed into two Gene Pulser Cuvetts (Bio-Rad) which then receive 8 ⁇ g of the reporter construct, and is electoporated using a Bio-Rad Gene Pulser at 210 volts and 960 ⁇ Faraday.
  • Bio-Rad Gene Pulser at 210 volts and 960 ⁇ Faraday.
  • the cells are washed and incubated with media containing charcoal stripped fetal bovine serum in the absence (blank) or presence (control) of 1 nM dihydrotestosterone (DHT; Sigma Chemical) and in the presence or absence of the standard anti-androgen bicalutamide or compounds of the present invention in concentrations ranging from 10 "10 to 10 "5 M (sample). Duplicates are used for each sample.
  • the spirocyclic compound dilutions are performed on a Biomek 2000 laboratory workstation. [0087] After 48 hours, a fraction of the supernatant is assayed for SEAP activity using the Phospha-Light Chemiluminescent Reporter Gene Assay System (Tropix, Inc). Viability of the remaining cells is determined using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS Assay, Promega).
  • a mix of a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) and an electron coupling reagent (phenazine methosulfate; PMS) are added to the cells.
  • MTS Oleen's reagent
  • PMS electron coupling reagent
  • % Control is referred as the effect of the tested compound compared to the maximal effect observed with the natural hormone, in this case DHT, and is calculated as:
  • the reporter plasmid utilized is comprised of the cDNA for the reporter SEAP protein, as described for the AR specific transactivation assay. Expression of the reporter SEAP protein is controlled by the mouse mammary tumor virus long terminal repeat (MMTV LTR) sequences that contains three hormone response elements (HREs) that can be regulated by both GR and PR see, e.g. G. Chalepakis et al., Cell, 53(3), 371 (1988). This plasmid is transfected into A549 cells, which expresses endogenous GR, to obtain a GR specific transactivation assay.
  • MMTV LTR mouse mammary tumor virus long terminal repeat
  • A549 cells are obtained from the American Type Culture Collection (Rockville, MD), and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Gibco). Determination of the GR specific antagonist activity of the spirocyclic compounds of the present invention is identical to that described for the AR specific transactivation assay, except that the DHT is replaced with 5 nM dexamethasone (Sigma Chemicals), a specific agonist for GR. Determination of the GR specific agonist activity of the spirocyclic compounds of the present invention is performed as described for the AR transactivation assay, wherein one measures the activation of the GR specific reporter system by the addition of a test compound, in the absence of a known GR specific agonists ligand.
  • the reporter plasmid utilized is comprised of the cDNA for the reporter SEAP protein, as described for the AR specific transactivation assay. Expression of the reporter SEAP protein is controlled by the mouse mammary tumor virus long terminal repeat (MMTV LTR) sequences that contains three hormone response elements (HREs) that can be regulated by both GR and PR. This plasmid is transfected into T47D, which expresses endogenous PR, to obtain a PR specific transactivation assay. T47D cells are obtained from the American Type Culture Collection (Rockville, MD), and maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco).
  • FBS fetal bovine serum
  • AR Binding Assay For the whole cell binding assay, human LNCaP cells (T877A mutant AR) or MDA 453 (wild type AR) in 96-well microtiter plates containing RPMI 1640 or DMEM supplemented with 10% charcoal stripped CA-FBS (Cocaleco Biologicals) respectively, are incubated at 37°C to remove any endogenous ligand that might be complexed with the receptor in the cells.
  • test compounds media containing 1 nM [ 3 H]-DHT and compounds of the invention in concentrations ranging from 10 "10 to 10 "5 M are added to the cells. Two replicates are used for each sample. After 4 hours at 37°C, cells are washed, harvested, and counted as described above.
  • the data is plotted as the amount of [ 3 H]-DHT (% of control in the absence of test compound) remaining over the range of the dose response curve for a given compound.
  • concentration of test compound that inhibited 50% of the amount of [ 3 H]-DHT bound in the absence of competing ligand is quantified (IC 5 0) after log-logit transformation.
  • the Ki values are determined by application of the Cheng-Prusoff equation to the IC 50 values, where:
  • IC 50 values are determined.
  • the IC 50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
  • the K d values for [ 3 H]-DHT for MDA 453 and LNCaP are 0.7 and 0.2 nM respectively.
  • Human Prostate Cell Proliferation Assay [0095] Compounds of the present invention are tested ("test compounds") on the proliferation of human prostate cancer cell lines. For that, MDA PCa2b cells, a cell line derived from the metastasis of a patient that failed castration, Navone et al., Clin.
  • the cells are treated in the absence (blank) or presence of 1 nM DHT (control) or with test compounds (sample) of the present invention in concentrations ranging from 10 "10 to 10 "5 M. Duplicates are used for each sample. The compound dilutions are performed on a Biomek 2000 laboratory work station. Seventy two hours later 0.44 uCi. of [ 3 H] -Thymidine (Amersham) is added per well and incubated for another 24 h followed by tripsinization, harvesting of the cells onto GF/B filters. Micro-scint PS are added to the filters before counting them on a Beckman TopCount. The % Inhibition was calculated as:
  • C2C12 Mouse Myoblast Transactivation Assay Two functional transactivation assays are developed to assess the efficacy of androgen agonists in a muscle cell background using a luciferase reporter.
  • the first assay (ARTA Stable 1) uses a cell line, Stable 1 (clone #72), which stably expresses the full length rat androgen receptor but requires the transient transfection of an enhancer/reporter. This cell line is derived from C2C12 mouse moyoblast cells.
  • the second assay (ARTA Stable 2) uses a cell line, Stable 2 (clone #133), derived from Stable 1 which stably expresses both rAR and the enhancer/luciferase reporter.
  • the enhancer/reporter construct used in this system is pGL3/2XDR- 1/luciferase.
  • 2XDR-1 was reported to be an AR specific response element in CV-I cells, Brown et. al. The Journal of Biological Chemistry 272, 8227-8235, (1997). It was developed by random mutagenesis of an AR/GR consensus enhancer sequence.
  • Stable 1 cells are plated in 96 well format at 6,000 cells/well in high glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029) containing 10% charcoal and dextran treated FBS (HyClone Cat. No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), IX MEM Na Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5X Antibiotic-Antimycotic, and 800 ⁇ g/ml Geneticin (Gibco BRL, Cat. No.: 10131-035). [0099] 2.
  • the cells are transfected with pGL3/2XDR-l/luciferase using LipofectAMINE PlusTM Reagent (Gibco BRL, Cat. No.: 10964-013).
  • LipofectAMINE PlusTM Reagent Gibco BRL, Cat. No.: 10964-013
  • 5 ng/well pGL3/2XDR-l/luciferase DNA and 50 ng/well Salmon Sperm DNA (as carrier) are diluted with 5 ⁇ l/well Opti-MEMem media (Gibco BRL, Cat. No.: 31985-070).
  • 0.5 ⁇ l/well Plus reagent is added.
  • ARTA stable 2 [00102] 1. Stable 2 cells are plated in 96 well format at 6,000 cells/well in high glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029) containing 10% charcoal and dextran treated EBS (HyClone Cat. No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat.
  • test compounds The ability of the spirocyclic compounds of the present invention to modulate the function of the AR is determined by testing said compounds in a proliferation assay using the androgen responsive murine breast cell line derived from the Shionogi tumor, Hiraoka et al, Cancer Res. , 47, 6560-6564 (1987).
  • Stable AR dependent clones of the parental Shionogi line are established by passing tumor fragments under the general procedures originally described in Tetuo, et. al., Cancer Res., 25, 1168-1175 (1965). From the above procedure, one stable line, SCl 14, is isolated, characterized, and utilized for the testing of example compounds.
  • SCl 14 cells are incubated with or without the test compounds for 72 hours and the amount of [ 3 H] -thymidine incorporated into DNA is quantified as a surrogate endpoint to assess the number of cells and therefore the proliferation rate as described in Suzuki et. al., J. Steroid Biochem. MoI. Biol. 37, 559-567 (1990).
  • the SCl 14 cell line is maintained in MEM containing 10 "8 M testosterone and 2% DCC- treated FCS.
  • cells are plated in 96- well microplates in the maintenance media and incubated at 37°C.
  • the medium is changed to serum free medium [Ham's F-12:MEM (1;1, v/v) containing 0.1% BSA] with (antagonist mode) or without (agonist mode) 10 "8 M testosterone and the test compounds of the present invention in concentrations ranging from 10 ⁇ 10 to 10 ⁇ 5 M.
  • Duplicates are used for each sample.
  • the spirocyclic compound dilutions are performed on a Biomek 2000 laboratory work station. Seventy two hours later 0.44 ⁇ Ci of [ 3 H]-Thymidine (Amersham) is added per well and incubated for another 2 hr followed by tripsinization, and harvesting of the cells onto GF/B filters. Micro-scint PS is added to the filters before counting them on a Beckman TopCount.
  • the % Inhibition is calculated as:
  • agonist mode % Control is referred as the effect of the tested compound compared to the maximal effect observed with the natural hormone, in this case DHT, and is calculated as:
  • the AP-I assay is a cell based luciferase reporter assay.
  • A549 cells which contain endogenous glucocorticoid receptor, are stably transfected with an AP- 1 DNA binding site attached to the luciferase gene. Cells are then grown in RPMI + 10% fetal calf serum (charcoal-treated) + Penicillin/Streptomycin with 0.5mg/ml geneticin. Cells are plated the day before the assay at approximately 40000 cells/well.
  • test compounds spirocyclic compounds of the present invention
  • test compounds dissolved in DMSO and added at varying concentrations
  • dexamethasome 100 nM in DMSO, positive control
  • the plates are then incubated for 7 hrs at 37°C after which 40 ⁇ l luciferase substrate reagent is added to each well.
  • Activity is measured by analysis in a luminometer as compared to control experiments treated with buffer or dexamethasome. Activity is designated as % inhibition of the reporter system as compared to the buffer control with 10 ng/ml PMA alone.
  • the control, dexamethasone, at a concentration of ⁇ 10 ⁇ M typically suppresses activity by 65%. Test compounds that demonstrate an inhibition of PMA induction of 50% or greater at a concentration of test compound of ⁇ 10 ⁇ M are deemed active.
  • Testosterone is converted to the more active form, dihydrotestosterone, (DHT), within the prostate by 5 ⁇ -reductase.
  • DHT dihydrotestosterone
  • Adrenal androgens also contribute about 20% of total DHT in the rat prostate, compared to 40% of that in 65-year-old men.
  • this is not a major pathway, since in both animals and humans, castration leads to almost complete involution of the prostate and seminal vesicles without concomitant adrenalectomy. Therefore, under normal conditions, the adrenals do not support significant growth of prostate tissues.
  • M. C. Luke and D. S. Coffey "The Physiology of Reproduction" ed. by E.
  • mice are dosed sub- cutaneously (s.c.) lmg/kg with Testosterone Proprionate (TP) in arachis oil vehicle and anti-androgen test compounds (compounds of the present invention) are dosed orally by gavage (p.o.) in dissolved/suspensions of 80% PEG 400 and 20% Tween 80 surfactant(PEGTW). Animals are dosed (v/w) at 0.5 ml of vehicle /10Og body weight. Experimental groups are as follows: 1. Control vehicle 2. Testosterone Propionate (TP) (3 mg/rat/day, subcutaneous) 3. TP plus Casodex (administered p.o.
  • test compound a compound of the present invention
  • TP s.c. as administered in group 2
  • test compound a compound of the present invention
  • TP s.c. as administered in group 2
  • the animals are sacrificed, and the ventral prostate weighed.
  • the sexual organs weights are first standardized as mg per 100 g of body weight, and the increase in organ weight induced by TP was considered as the maximum increase (100%).
  • ANOVA followed by one-tailed Student or Fischer's exact test is used for statistical analysis.
  • the gain and loss of sexual organ weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration. See Y. Okuda et al., /. Urol, 145, 188-191 (1991), the disclosure of which is herein incorporated by reference.
  • organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonist.
  • replacement of exogenous androgens increases seminal vesicles (SV) and the ventral prostate (VP) in a dose dependent manner.
  • the maximum increase in organ weight is 4 to 5-fold when dosing 3 mg/rat/day of testosterone (T) or 1 mg/rat/day of testosterone propionate (TP) for 3 days.
  • the EC 50 of T and TP are about 1 mg and 0.03 mg, respectively.
  • the increase in the weight of the VP and SV also correlates with the increase in the serum T and DHT concentration.
  • TP showed about 10-30-fold higher potency than free T.
  • a known AR antagonist (Casodex) is also administered simultaneously with 0.1 mg of TP (ED 8 o), inhibiting the testosterone-mediated increase in the weights of the VP and SV in a dose dependent manner.
  • the antagonist effects are similar when dosing orally or subcutaneously.
  • Compounds of the invention also exhibit AR antagonist activity by suppressing the testosterone-mediated increase in the weights of VP and SV.
  • the rats are divided into groups and treated daily for 7 to 14 days with one of the following: 1. Control vehicle 2. Testosterone Propionate (TP) (3 mg/rat/day, subcutaneous) 3. TP plus Casodex (administered p.o. in PEGTW, QD), a recognized antiandrogen, as a reference compound. 4. To demonstrate antagonist activity, a compound of the present invention (“test compound”) is administered (p.o. in PEGTW, QD) with TP (s.c. as administered in group 2) in a range of doses. 5. To demonstrate agonist activity, a compound of the present invention (“test compound”) is administered alone (p.o. in PEGTW, QD) in a range of doses.
  • the animals are sacrificed by carbon dioxide, and the levator ani, seminal vesicle and ventral prostate weighed.
  • the levator ani muscle and sexual organ weights are first standardized as mg per 100 g of body weight, and the increase in organ weight induced by TP is considered as the maximum increase (100%).
  • Super- anova one factor is used for statistical analysis.
  • the gain and loss of sexual organ weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration. See Y. Okuda et al., J. Urol, 145, 188-191 (1991), the disclosure of which is herein incorporated by reference.
  • organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonist.
  • replacement of exogenous androgens increase levator ani, seminal vesicles (SV) and prostate in a dose dependent manner.
  • the maximum increase in organ weight is 4 to 5-fold when dosing 3 mg/rat/day of testosterone (T) or 1 mg/rat/day of testosterone propionate (TP) for 3 days.
  • the EC 50 of T and TP are about 1 mg and 0.03 mg, respectively.
  • the increase in the weight of the VP and SV also correlates with the increase in the serum T and DHT concentration.
  • T shows 5-times higher serum concentrations of T and DHT at 2 hours after subcutaneous injection than that of TP, thereafter, these high levels decline very rapidly.
  • serum concentrations of T and DHT in TP-treated animals are fairly consistent during the 24 hours, and therefore, TP shows about 10-30-fold higher potency than free T.
  • MDA PCa2b Human Prostate Zenograft Assay [00119] In Vivo Antitumor Testing: MDA-PCa-2b human prostate tumors are maintained in Balb/c nu/nu nude mice. Tumors are propagated as subcutaneous transplants in adult male nude mice (4-6 weeks old) using tumor fragments obtained from donor mice. Tumor passage occurs every 5-6 weeks. [00120] For antitumor efficacy trial, the required number of animals needed to detect a meaningful response are pooled at the start of the experiment and each is given a subcutaneous implant of a tumor fragment (-50 mg) with a 13-gauge trocar.
  • Tumors are allowed to grow to approximately 100-200 mg (tumors outside the range were excluded) and animals are evenly distributed to various treatment and control groups. Treatment of each animal is based on individual body weight. Treated animals are checked daily for treatment related toxicity/mortality. Each group of animals is weighed before the initiation of treatment (Wt 1 ) and then again following the last treatment dose (Wt 2 ). The difference in body weight (Wt 2 -Wt 1 ) provides a measure of treatment-related toxicity. ⁇ [00121] Tumor response is determined by measurement of tumors with a caliper twice a week, until the tumors reach a predetermined "target" size of 0.5 gm.
  • Tumor response end-point is expressed in terms of tumor growth inhibition (%T/C), defined as the ratio of median tumor weights of the treated tumors (T) to that of the control group (C).
  • %T/C tumor growth inhibition
  • Log cell kill (T-C) ⁇ (3.32 x TVDT).
  • Statistical evaluations of data are performed using Gehan's generalized Wilcoxon test.
  • Dunning Prostate Tumor is a spontaneously derived, well differentiated androgen responsive adenocarcinoma of the prostate (Smolev JK, Heston WD, Scott WW, and Coffey DS, Cancer Treat Rep. 61, 273-287 (1977)).
  • the growth of the R3327H subline has been selected for its highly androgen-dependent and reproducible growth in intact male rats. Therefore, this model and other sublines of this tumor have been widely used to evaluate in vivo antitumor activities of antiandrogens such as flutamide and bacilutamide/Casodex (Maucher A., and von Angerer, J. Cancer Res. Clin.
  • the Dunning tumor pieces (about 4 x 4 mm) are transplanted subcutaneously to the flank of mature male Copenhagen rats (6-7 weeks old, Harlan-Sprague Dawley, Indianapolis, MD). About 6 weeks after the implantation, the animals with tumors of measurable size (about 80-120 mm 2 ) are randomized into treatment groups (8-10 rats/group) and the treatments are initiated. One group of the rats is castrated to serve as the negative control of tumor growth.
  • mice are treated daily with compounds of the current invention, standard antiandrogens such as bacilutamide or vehicle (control) for an average of 10 to 14 weeks.
  • Test compounds are dissolved in a vehicle of (2.5 ml/kg of body weight) 10% polyethylene glycol and 0.05% Tween-80 surfactant in 1% carboxymethyl cellulose, PEG/CMC, (Sigma, St Louis, MO).
  • Typical therapeutic experiments would include three groups of three escalating doses for each standard or test compound (in a range of 300-3 mg/kg).
  • Tumors in the vehicle (control) group reach a size of 1500 to 2500 mm 3 , whereas the castrated animal group typically shows tumor stasis over the 14 weeks of observation.
  • Mature Rat Prostate Weight Assay [00127] The activity of compounds of the present invention are investigated in a mature male rat model, which is a variation of the Levator ani & wet prostate weight assay described above.
  • the above in vivo assays are recognized assays for determining the anabolic effects in muscle and sustaining effects in sex organs for a given compound, as described in L. G. Hershberger et al, 83 Proc. Soc. Expt. Biol. Med., 175 (1953); B. L. Beyler et al, "Methods for evaluating anabolic and catabolic agents in laboratory animals", 23 J. Amer. Med. Women's Ass., 708 (1968); H.
  • T serum testosterone
  • LH pituitary luteinizing hormone
  • FSH follicle stimulating hormone
  • DHT dihydrotestosterone
  • Testosterone production in the Leydig cells of the testis is controlled by the level of circulating LH released from the pituitary gland. LH levels are themselves controlled by the level of LHRH produced in the hypothalmic region. Testosterone levels in the blood serve to inhibit the secretion of LHRH and subsequently reduce levels of LH and ultimately the levels of circulating testosterone levels.
  • test compounds By measuring blood levels of LH as they are effected by compounds of the present invention ("test compounds”), it is possible to determine the level of agonist or antagonist activity of said compounds at the hypothalamic axis of this endocrine cycle.
  • Bicalutamide (Casodex, a recognized antiandrogen, as a reference compound) or a compound of the present invention, p.o. in PEGTW QD. (in a range of doses).
  • the animals are sacrificed, and the ventral prostate, the seminal vesicles, and the levator ani are removed surgically and weighed.
  • the organs weights are first standardized as mg per 100 g of body weight, and expressed as a percentage of the value of the respective organ in the intact group.
  • Rat luteinizing hormone is quantitatively determined with the Biotrak [125 I] kit (Amersham Pharmacia Biotek), following the manufacturer directions. The assay is based on the competition by the LH present in the serum of the binding of [ 125 I] rLH to an Amerlex-M bead/antibody suspension. The radioactivity that remains after incubation with the serum and subsequent washes is extrapolated into a standard curve to obtain a reading in ng/ml.
  • the gain and loss of sexual organ and levator ani weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration, see Y. Okuda et al, J.
  • CWR22 Human Prostate Zenograft Assay [00134] In Vivo Antitumor Testing: CWR22 human prostate tumors are maintained in Balb/c nu/nu nude mice. Tumors are propagated as subcutaneous transplants in adult male nude mice (4-6 weeks old) using tumor fragments obtained from donor mice. Tumor passage occurs every 5-6 weeks. [00135] For antitumor efficacy trial, the required number of animals needed to detect a meaningful response are pooled at the start of the experiment and each is given a subcutaneous implant of a tumor fragment (-50 mg) with a 13-gauge trocar. Tumors are allowed to grow to approx. 100-200 mg (tumors outside the range were excluded) and animals are evenly distributed to various treatment and control groups.
  • Treatment of each animal is based on individual body weight. Treated animals are checked daily for treatment related toxicity/mortality. Each group of animals is weighed before the initiation of treatment (Wt 1 ) and then again following the last treatment dose (Wt 2 ). The difference in body weight (Wt 2 -Wt 1 ) provides a measure of treatment-related toxicity.
  • Tumor response end-point is expressed in terms of tumor growth inhibition (%T/C), defined as the ratio of median tumor weights of the treated tumors (T) to that of the control group (C).
  • %T/C tumor growth inhibition
  • Log cell kill (T-C) ⁇ (3.32 x TVDT)
  • Statistical evaluations of data are performed using Gehan's generalized Wilcoxon test.
  • preferred compounds of the present invention such as particular compounds disclosed in the following examples, have been identified to modulate the activity of one or more of nuclear hormone receptors.
  • Exemplary values for compounds that inhibit activity in one or more assays include concentrations equivalent to or more potent than 20 ⁇ M, preferably 10 ⁇ M, and more preferably 1 ⁇ M.
  • Example 1 The crude product was purified by flash chromatography on SiO 2 using gradient elution (0 - 10 - 20% acetone in chloroform) to give 0.134 g of Example 1 as a yellow solid.
  • HPLC 100% at 2.513 min (retention time) (YMC S5 ODS column 4.6 x 50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 320.23 [M+H] + .

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Abstract

L'invention porte sur des composés spirocycliques, des procédés d'utilisation de ces composés spirocycliques dans le traitement des conditions associées au récepteur de l'hormone nucléaire telles que le cancer et les troubles immunitaires, ainsi que sur des compositions pharmaceutiques contenant ces composés.
PCT/US2005/021823 2004-06-21 2005-06-21 Composes spirocycliques utiles en tant que modulateurs de la fonction du recepteur de l'hormone nucleaire Ceased WO2006002102A2 (fr)

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US10806697B2 (en) 2012-12-21 2020-10-20 Therapeuticsmd, Inc. Vaginal inserted estradiol pharmaceutical compositions and methods
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